Patterns of selective constraints in noncoding DNA of rice

<p>Abstract</p> <p>Background</p> <p>Several studies have investigated the relationships between selective constraints in introns and their length, GC content and location within genes. To date, however, no such investigation has been done in plants. Studies of selective constraints in noncoding DNA have generally involved interspecific comparisons, under the assumption of the same selective pressures acting in each lineage. Such comparisons are limited to cases in which the noncoding sequences are not too strongly diverged so that reliable sequence alignments can be obtained. Here, we investigate selective constraints in a recent segmental duplication that includes 605 paralogous intron pairs that occurred about 7 million years ago in rice (<it>O. sativa</it>).</p> <p>Results</p> <p>Our principal findings are: (1) intronic divergence is negatively correlated with intron length, a pattern that has previously been described in <it>Drosophila </it>and mammals; (2) there is a signature of strong purifying selection at splice control sites; (3) first introns are significantly longer and have a higher GC content than other introns; (4) the divergences of first and non-first introns are not significantly different from one another, a pattern that differs from <it>Drosophila </it>and mammals; and (5) short introns are more diverged than four-fold degenerate sites suggesting that selection reduces divergence at four-fold sites.</p> <p>Conclusion</p> <p>Our observation of stronger selective constraints in long introns suggests that functional elements subject to purifying selection may be concentrated within long introns. Our results are consistent with the presence of strong purifying selection at splicing control sites. Selective constraints are not significantly stronger in first introns of rice, as they are in other species.</p

Selection and mutation on microRNA target sequences during rice evolution

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) posttranscriptionally down-regulate gene expression by binding target mRNAs. Analysis of the evolution of miRNA binding sites is helpful in understanding the co-evolution between miRNAs and their targets. To understand this process in plants a comparative analysis of miRNA-targeted duplicated gene pairs derived from a well-documented whole genome duplication (WGD) event in combination with a population genetics study of six experimentally validated miRNA binding sites in rice (<it>O. sativa</it>) was carried out.</p> <p>Results</p> <p>Of the 1,331 pairs of duplicate genes from the WGD, 41 genes (29 pairs) were computationally predicted to be miRNA targets. Sequence substitution analysis indicated that the synonymous substitution rate was significantly lower in the miRNA binding sites than their 5' and 3' flanking regions. Of the 29 duplicated gene pairs, 17 have only one paralog been targeted by a miRNA. This could be due to either gain of a miRNA binding site after the WGD or because one of the duplicated genes has escaped from being a miRNA target after the WGD (loss of miRNA binding site). These possibilities were distinguished by separating miRNAs conserved in both dicots and monocot plants from rice-specific miRNAs and by phylogenetic analysis of miRNA target gene families. The gain/loss rate of miRNA binding sites was estimated to be 3.0 × 10^-9gain/loss per year. Most (70.6%) of the gains/losses were due to nucleotide mutation. By analysis of cultivated (<it>O. sativa</it>; <it>n </it>= 30) and wild (<it>O. rufipogon</it>; <it>n </it>= 15) rice populations, no segregating site was observed in six miRNA binding sites whereas 0.12–0.20 SNPs per 21-nt or 1.53–1.80 × 10^-3of the average pairwise nucleotide diversity (π) were found in their flanking regions.</p> <p>Conclusion</p> <p>Both molecular evolution and population genetics support the hypothesis that conservation of miRNA binding sites is maintained by purifying selection through elimination of deleterious alleles. Nucleotide mutations play a major role in the gain/loss of miRNA binding sites during evolution.</p

Research on the End Surface Dent of the Main Shaft Forging

In the process of the stretching of the shaft forgings, if the process parameters are not properly selected, the end-face dent will take place. The end-face dent affects the performance of large forgings and leads to much material wasting. Finite element method was employed to perform numerical simulation of the stretching of a main shaft with an upper flat anvil and a lower V-shaped anvil. The orthogonal test table was designed by selecting the anvil width, the Reduction ratio and the feed as influencing factors. Accordingly, simulations were carried out to solve the end-face dent values under different parameter combinations. The analysis showed that the optimal parameter combination gave an anvil width ratio of 0.75, a Reduction ratio of 0.2, and an initial feed of 300 mm. Through extremum difference analysis, it was found that among the three factors are the feed, the reduction ratio, and the anvil width ratio in the decreasing order of the influence on the end- face dent. Comprehensive analysis showed that when the anvil is relatively narrow, increasing the relative feed can reduce the end-surface dent remarkably. It is advisable that during the stretching of shaft forgings with a flat upper anvil and a V-shaped lower anvil, the combination of the anvil width ratio of 0.75, the reduction ratio of 0.2, and increasing the feed can reduce the end-face dent, thereby reducing the end cutting and saving material costs

Optimization of embryogenic-callus induction and embryogenesis of Glycyrrhiza glabra

Glabridin is a major biologically active flavonoid isolated specifically from the root of Glycyrrhiza glabra, which has many pharmacological activities. The production of the wild G. glabra was sharply decreased due to immoderate and ruinous utilization. In vitro regeneration via somatic embryogenesis is important for clonal propagation and genetic transformation. In this paper, factors affecting the embryogenic calli and embryo induction, maintenance and multiplication of G. glabra are assessed. The results showed that the explants of hypocotyl give the highest calli formation frequency of 93.3% on Murashige and Skoog (MS) medium containing 2.0 mg/L 6-benzylaminopurine (6-BA) and 0.5 mg/L 2,4- dichlorophenoxyacetic acid (2,4-D). The maximum efficiency of embryo were obtained on MS medium with 0.5 mg/L 6-BA + 0.5 mg/L kinetin zeatin (KT) + 0.1 mg/L indole-3-butyric acid (IBA); the embryos could develop further on medium with 1000 mg/L malt extract (ME). The occurrence of the embryogenic calli and proglobular embryo were studied by histological section, indicating the single cell origin of the embryogensis of G. glabra. With the protocol reported herein, some green embryo-like cultures were obtained, from which shoots were successfully regenerated in the germinated medium after 10 months of subculture.Keywords: Glycyrrhiza glabra L., callus induction, embryogenesis, cell culture, histological sectionAfrican Journal of Biotechnology Vol. 9(36), pp. 5823-5829, 6 September, 201

Characterization of Small Interfering RNAs Derived from the Geminivirus/Betasatellite Complex Using Deep Sequencing

BACKGROUND: Small RNA (sRNA)-guided RNA silencing is a critical antiviral defense mechanism employed by a variety of eukaryotic organisms. Although the induction of RNA silencing by bipartite and monopartite begomoviruses has been described in plants, the nature of begomovirus/betasatellite complexes remains undefined. METHODOLOGY/PRINCIPAL FINDINGS: Solanum lycopersicum plant leaves systemically infected with Tomato yellow leaf curl China virus (TYLCCNV) alone or together with its associated betasatellite (TYLCCNB), and Nicotiana benthamiana plant leaves systemically infected with TYLCCNV alone, or together with TYLCCNB or with mutant TYLCCNB were harvested for RNA extraction; sRNA cDNA libraries were then constructed and submitted to Solexa-based deep sequencing. Both sense and anti-sense TYLCCNV and TYLCCNB-derived sRNAs (V-sRNAs and S-sRNAs) accumulated preferentially as 22 nucleotide species in infected S. lycopersicum and N. benthamiana plants. High resolution mapping of V-sRNAs and S-sRNAs revealed heterogeneous distribution of V-sRNA and S-sRNA sequences across the TYLCCNV and TYLCCNB genomes. In TYLCCNV-infected S. lycopersicum or N. benthamiana and TYLCCNV and βC1-mutant TYLCCNB co-infected N. benthamiana plants, the primary TYLCCNV targets were AV2 and the 5' terminus of AV1. In TYLCCNV and betasatellite-infected plants, the number of V-sRNAs targeting this region decreased and the production of V-sRNAs increased corresponding to the overlapping regions of AC2 and AC3, as well as the 3' terminal of AC1. βC1 is the primary determinant mediating symptom induction and also the primary silencing target of the TYLCCNB genome even in its mutated form. CONCLUSIONS/SIGNIFICANCE: We report the first high-resolution sRNA map for a monopartite begomovirus and its associated betasatellite using Solexa-based deep sequencing. Our results suggest that viral transcript might act as RDR substrates resulting in dsRNA and secondary siRNA production. In addition, the betasatellite affected the amount of V-sRNAs detected in S. lycopersicum and N. benthamiana plants

Identification of wounding and topping responsive small RNAs in tobacco (Nicotiana tabacum)

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are two major classes of small RNAs. They play important regulatory roles in plants and animals by regulating transcription, stability and/or translation of target genes in a sequence-complementary dependent manner. Over 4,000 miRNAs and several classes of siRNAs have been identified in plants, but in tobacco only computational prediction has been performed and no tobacco-specific miRNA has been experimentally identified. Wounding is believed to induce defensive response in tobacco, but the mechanism responsible for this response is yet to be uncovered.</p> <p>Results</p> <p>To get insight into the role of small RNAs in damage-induced responses, we sequenced and analysed small RNA populations in roots and leaves from wounding or topping treated tobacco plants. In addition to confirmation of expression of 27 known miRNA families, we identified 59 novel tobacco-specific miRNA members of 38 families and a large number of loci generating phased 21- or 24-nt small RNAs (including ta-siRNAs). A number of miRNAs and phased small RNAs were found to be responsive to wounding or topping treatment. Targets of small RNAs were further surveyed by degradome sequencing.</p> <p>Conclusions</p> <p>The expression changes of miRNAs and phased small RNAs responsive to wounding or topping and identification of defense related targets for these small RNAs suggest that the inducible defense response in tobacco might be controlled by pathways involving small RNAs.</p

Post-Domestication Selection in the Maize Starch Pathway

Modern crops have usually experienced domestication selection and subsequent genetic improvement (post-domestication selection). Chinese waxy maize, which originated from non-glutinous domesticated maize (Zea mays ssp. mays), provides a unique model for investigating the post-domestication selection of maize. In this study, the genetic diversity of six key genes in the starch pathway was investigated in a glutinous population that included 55 Chinese waxy accessions, and a selective bottleneck that resulted in apparent reductions in diversity in Chinese waxy maize was observed. Significant positive selection in waxy (wx) but not amylose extender1 (ae1) was detected in the glutinous population, in complete contrast to the findings in non-glutinous maize, which indicated a shift in the selection target from ae1 to wx during the improvement of Chinese waxy maize. Our results suggest that an agronomic trait can be quickly improved into a target trait with changes in the selection target among genes in a crop pathway

Sequence variation and selection of small RNAs in domesticated rice

<p>Abstract</p> <p>Background</p> <p>Endogenous non-coding small RNAs (21-24 nt) play an important role in post-transcriptional gene regulation in plants. Domestication selection is the most important evolutionary force in shaping crop genomes. The extent of polymorphism at small RNA loci in domesticated rice and whether small RNA loci are targets of domestication selection have not yet been determined.</p> <p>Results</p> <p>A polymorphism survey of 94 small RNA loci (88 <it>MIRNAs</it>, four <it>TAS3 </it>loci and two miRNA-like long hairpins) was conducted in domesticated rice, generating 2 Mb of sequence data. Many mutations (substitution or insertion/deletion) were observed at small RNA loci in domesticated rice, e.g. 12 mutation sites were observed in the mature miRNA sequences of 11 <it>MIRNAs </it>(12.5% of the investigated <it>MIRNAs</it>). Several small RNA loci showed significant signals for positive selection and/or potential domestication selection.</p> <p>Conclusions</p> <p>Sequence variation at miRNAs and other small RNAs is higher than expected in domesticated rice. Like protein-coding genes, non-coding small RNA loci could be targets of domestication selection and play an important role in rice domestication and improvement.</p

TcMYC2a, a Basic Helix–Loop–Helix Transcription Factor, Transduces JA-Signals and Regulates Taxol Biosynthesis in Taxus chinensis

The multitherapeutic taxol, which can be obtained from Taxus spp., is the most widely used anticancer drug. Taxol biosynthesis is significantly regulated by jasmonate acid (JA), one of the most important endogenous hormones in land plants. Nevertheless, the JA-inducing mechanism remains poorly understood. MYC2 is one of the key regulators of JA signal transfer and the biosynthesis of various secondary metabolites. Here, TcMYC2a was identified to contain a basic helix–loop–helix (bHLH)-leucine zipper domain, a bHLH-MYC_N domain, and a BIF/ACT-like domain. TcMYC2a was also found to bind with TcJAZ3 in yeast, which was a homolog of Arabidopsis JASMONATE ZIM-domain JAZ proteins, indicating that TcMYC2a had a similar function to AtMYC2 of JA signal transduction. TcMYC2a was able to affect the expression of GUS reporter gene by binding with the T/G-box, G-box, and E-box, which were the key cis-elements of TASY and TcERF12/15 promoter. TcMYC2a overexpression also led to significantly increased expression of TASY, tat, dbtnbt, t13h, and t5h genes. Additionally, TcERF15, which played the positive role to regulate tasy gene, was up-regulated by TcMYC2a. All these results revealed that TcMYC2a can regulate taxol biosynthesis either directly or via ERF regulators depending on JA signaling transduction